Chitose Ryo, Watanabe Atsushi, Asano Masato, Hanashima Akira, Sasano Kouhei, Bao Yulong, Maruyama Koscak, Kimura Sumiko
Department of Biology, Graduate School of Science, Chiba University, Chiba 263-8522, Japan.
J Biomed Biotechnol. 2010;2010:108495. doi: 10.1155/2010/108495. Epub 2010 May 12.
Nebulin is about 800 kDa filamentous protein that binds the entire thin filament of vertebrate skeletal muscle sarcomeres. Nebulin cannot be isolated from muscle except in a completely denatured form by direct solubilization of myofibrils with SDS because nebulin is hardly soluble under salt conditions. In the present study, nebulin was solubilized by a salt solution containing 1 M urea and purified by DEAE-Toyopearl column chromatography via 4 M urea elution. Rotary-shadowed images of nebulin showed entangled knit-like particles, about 20 nm in diameter. The purified nebulin bound to actin filaments to form loose bundles. Nebulin was confirmed to bind actin, alpha-actinin, beta-actinin, and tropomodulin, but not troponin or tropomyosin. The data shows that full-length nebulin can be also obtained in a functional and presumably native form, verified by data from experiments using recombinant subfragments.
伴肌动蛋白是一种约800 kDa的丝状蛋白，它与脊椎动物骨骼肌肌节的整个细肌丝结合。伴肌动蛋白很难在盐条件下溶解，因此除非通过用SDS直接溶解肌原纤维使其完全变性，否则无法从肌肉中分离出来。在本研究中，伴肌动蛋白通过含有1 M尿素的盐溶液溶解，并通过DEAE - 琼脂糖凝胶柱色谱法经4 M尿素洗脱进行纯化。伴肌动蛋白的旋转阴影图像显示出直径约20 nm的缠结针织状颗粒。纯化的伴肌动蛋白与肌动蛋白丝结合形成松散的束。已证实伴肌动蛋白与肌动蛋白、α - 辅肌动蛋白、β - 辅肌动蛋白和原肌球蛋白结合，但不与肌钙蛋白或原肌球蛋白结合。数据表明，通过使用重组亚片段的实验数据验证，全长伴肌动蛋白也可以以功能形式且可能是天然形式获得。
